This invention relates to nucleic acid and amino acid sequences of a novel inhibitor of programmed cell death, the protein Aven, and to the use of these sequences in the diagnosis, prevention, and treatment of diseases associated with decreased or increased apoptosis.
Normal development, growth, and homeostasis in multi-cellular organisms require a careful balance between the production and destruction of cells in tissues throughout the body. Cell division is a carefully coordinated process with numerous checkpoints and control mechanisms. These mechanisms are designed to regulate DNA replication and to prevent inappropriate or excessive proliferation. In contrast, apoptosis is the genetically controlled process by which unneeded or damaged cells can be eliminated without causing the tissue destruction and inflammatory responses that are often associated with acute injury and necrosis.
The term xe2x80x9capoptosisxe2x80x9d was first used to describe the morphological changes that characterize cells undergoing programmed cell death. Apoptotic cells have a shrunken appearance with an altered membrane lipid content and highly condensed nuclei. Apoptotic cells are rapidly phagocytosed by neighboring cells or macrophages without leaking their potentially damaging contents into the surrounding tissue or triggering an inflammatory response.
The processes and mechanisms regulating apoptosis are highly conserved throughout the phylogenetic tree, and much of our current knowledge about apoptosis is derived from studies of the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. Aberrations in apoptosis regulation have recently been recognized as significant factors in the pathogenesis of human disease. For example, inappropriate cell survival can cause or contribute to many diseases such as cancer, autoimmune diseases, and inflammatory diseases. In contrast, increased apoptosis can cause immunodeficiency diseases such as AIDS, neurodegenerative disorders, and myelodysplastic syndromes.
A variety of ligands and their cellular receptors, enzymes, tumor suppressors, viral gene products, pharmacological agents, and inorganic ions have important positive or negative roles in regulating and implementing the apoptotic destruction of a cell. Although some specific components of the apoptotic pathway have been identified and characterized, many interactions between the proteins involved are undefined, leaving major aspects of the pathway unknown. Despite the identification of genes necessary for cell death and the ability to regulate apoptosis by known genes, the essential biochemical events in apoptotic death remain largely unknown.
The consistency of the morphologic and biochemical patterns defined as apoptosis within different cell types and species, during normal development and as a response to external stimuli arc consistent with a common cause of cellular mortality. The thesis is supported by the concept of an endogenous program responsible for cells death and the presence of gene products which are positive and negative regulators of apoptosis. The best studied negative regulator of apoptosis is the Bcl-2 proto-oncogene product. It provides the strongest evidence for a sharedmammalian pathway of death by its ability to block a wide variety of cell death models.
The Bcl-2 proto-oncogene is rather unique among cellular genes in its ability to block apoptotic deaths in multiple contexts. Overexpression of Bcl-2 in transgenic models leads to accumulation of cells due to evasion of normal cell death mechanisms. Induction of apoptosis by diverse stimuli, such as radiation, hyperthermia, growth factor withdrawal, glucocorticoids and multiple classes of chemotherapeutic agents is inhibited by Bcl-2 in vitro models. These effects are proportional to the level of Bcl-2 expression. Additionally, the endogenous pattern of Bcl-2 expression is indicative of a role in the regulation of cell survival in vivo. The Bcl-2 protein seems likely to function as an antagonist of a central mechanism operative in cell death.
The protein encoded by the Bcl-2 proto-oncogene has been reported to be capable of inhibiting apoptosis in many hematopoietic cell systems. The proto-oncogene Bcl-2 was isolated and characterized as a result of its frequent translocation adjacent to the immunoglobulin heavy chain enhancer in the t(14;18) chromosome translocation present in more than 80% of human follicular lymphomas. These neoplasias are characterized by an accumulation of mature resting B cells presumed to result from a block of apoptosis which would normally cause turnover of these cells. Transgenic mice expressing Bcl-2 under the control of the Excexc enhancer similarly develop follicular lymphomas.
The Bcl-2 protein is a 26 kDa membrane-associated cytoplasmic protein. Unlike many other proto-onocogene products, the Bcl-2 protein apparently functions, at least in part, by enhancing the survival of hematopoietic cells of T and B origins rather than by directly promoting proliferation of these cell types. The capacity of Bcl-2 to enhance cell survival is related to its ability to inhibit apoptosis initiated by several factors, such as cytokine deprivation, radiation exposure, glucocorticoid treatment, and administration of anti-CD-3 antibody. Upregulation of Bcl-2 expression also inhibits apoptosis of EBV-infected B-cell lines. The expression of Bcl-2 has also been shown to block apoptosis resulting from expression of the positive cell growth regulatory proto-oncogene, c-myc, in the absence of serum or growth factors.
Within vertebrates, Bcl-2 is the best understood gene in a cell death pathway and functions as a cell death repressor. Other proteins which interact with and/or are structurally related to the Bcl-2 gene product have also been identified, such as, for example, Bcl-xL and Bcl-xS. The family of Bcl-2 related proteins also includes the nematode protein CED-9 and two DNA virus proteins, LMW5-HL and BHRF-1 of the Epstein Barr Virus. Thus, a family of Bcl-2 like genes exists and evidence indicates that they participate in regulating cell death.
The family of Bcl-2 related proteins has been noted to have homology that is principally, but not exclusively, clustered within two conserved regions entitled Bcl-homology 1 and 2 (BH1 and BH2). This includes Bax, Bcl-XL, Mcl-1 and Al, and several open reading frames in DNA viruses including BHRF-1 of Epstein-Barr virus and LMW5-HL of African swine fever virus.
It has been discovered that Bcl-2 also associates in vivo with a 21 kDa protein partner called Bax. Bax shows extensive amino acid homology with Bcl-2 and forms homodimers with itself and heterodimers with Bcl-2 in vivo. Bax is encoded by 6 exons and demonstrates a complex pattern of alternative RNA splicing that predicts a 21 KDa membrane (xcex1) and three forms (xcex2, xcex3, and xcfx89)) of cytosolic protein. Bcl-2 and Bax have biochemical functions that are yet to be delineated but may also modulate cell death/survival through heterodimerization. When Bax predominates and a substantial percentage of Bax is present as homomultimers (e.g., homodimers) and/or free (unbound) Bax monomer or other activated form or complex, programmed cell death is accelerated and the death repressor activity of Bcl-2 is countered. In many cell types when in excess, Bax counters the ability of Bcl-2 to repress cell death. It was unexpected to find that Bax shares extensive homology with Bcl-2, especially within two highly conserved domains. These domains are also the most highly conserved regions of human, mouse, and chicken Bcl-2. These domains are also conserved in an open reading frame BHRF-1 within Epstein-Barr virus and Mcl-1, a gene recently isolated from a myeloid leukemia cell line following induction with phorbol ester.
The Bcl-x gene was identified by low-stringency hybridization using a Bcl-2 polynucleotide probe and encodes two proteins, Bcl-xL and Bcl-xS, via alternative RNA splicing. The Bcl-xL cDNA encodes a polypeptide of 233 amino acids with similar domains to those of Bcl-2. The Bcl-xS cDNA encodes a polypeptide of 170 amino acids in which the region of highest homology to Bcl-2 has been deleted. When the ability of these two proteins to regulate apoptotic cell death was compared, it was found that Bcl-xL rendered cells resistant to apoptotic cell death induced by growth factor deprivation, whereas Bcl-xS could prevent overexpression of Bcl-2 from inducing resistance to apoptotic cell death. Thus, Bcl-xL can serve as an inhibitor of apoptotic cell death in a variety of cell lines, whereas Bcl-xS inhibits the ability of Bcl-2 to inhibit cell death and can make cells more susceptible to apoptotic cell death.
Many pathological conditions result, at least in part, from aberrant control of cell proliferation, differentiation and/or apoptosis. For example, neoplasia is characterized by a clonally derived cell population which has a diminished capacity for responding to normal cell proliferation control signals. Oncogenic transformation of cells leads to a number of changes in cellular metabolism, physiology, and morphology. One characteristic alteration of oncogenically transformed cells is a loss of responsiveness to constraints on cell proliferation and differentiation normally imposed by the appropriate expression of cell growth regulatory genes.
The precise molecular pathways and secondary changes leading to malignant transformation for many cell types are not clear. However, the characteristic translocation of the apoptosis-associated Bcl-2 gene to the immunoglobulin heavy chain locus t(14;18) in more than 80 percent of human follicular B cell lymphomas and 20 percent of diffuse lymphomas and the neoplastic follicular lymphoproliferation present in transgenic mice expressing high levels of Bcl-2 indicates that the Bcl-2 gene likely is causally involved in neoplastic diseases and other pathological conditions resulting from abnormal apoptosis, cell proliferation, and differentiation. Thus, it is desirable to identify agents which can modify the activity(ies) of Bcl-2-related proteins so as to modulate apoptosis, cell proliferation, and differentiation for therapeutic or prophylactic benefit. Further, such agents can serve as commercial research reagents for control of apoptosis cell proliferation, and differentiation in experimental applications, and/or for controlled proliferation and differentiation of predetermined hematopoietic stem cell populations in vitro, in ex vivo therapy, or in vivo.
Despite progress in developing a more defined model of the molecular mechanisms underlying the transformed phenotype and neoplasia, few significant therapeutic methods applicable to treating cancer beyond conventional chemotherapy have resulted. Such Bcl-2-related protein modulating agents can provide novel chemotherapeutic agents for treatment of neoplasia, lymphoproliferative conditions, arthritis, inflammation, autoimmune diseases, and the like. The present invention fulfills these and other needs.
While identifying the Bcl-2 cell death pathway is significant, a way of regulating the Bcl-2 pathway has not been achieved. The ability to down-regulate the cell death repressing effect of Bcl-2 and/or up-regulate the cell death promoting activity of Bax would be advantageous in cancer therapy, in controlling hyperplasia such as benign prostatic hypertrophy (BPH) and eliminating self reactive clones in autoimmunity by favoring death effector molecules. Up-regulating the effect of Bcl-2 and favoring death repressor molecules would be beneficial in the treatment and diagnosis of immunodeficiency diseases, including AIDS, senescence, neurodegenerative disease, ischemic cell death, wound-healing, and the like.
The discovery of polynucleotides encoding a Bcl-xL interacting factor (Aven), and the molecules themselves, provides a means to investigate the regulation of programmed cell death and apoptosis. Discovery of molecules related to apoptosis regulator proteins satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the detection, prevention, and treatment of cancer, autoimmune diseases, lymphoproliferative disorders, atherosclerosis, AIDS, immunodeficiency diseases, ischemic injuries, neurodegenerative diseases, osteoporosis, myelodysplastic syndromes, toxin-induced diseases, and viral infections.
The present invention relates to the discovery that Bcl-xL interacts with a 55 kDa protein called Aven (also known as Bif-1 (Bcl-xL, Interacting Factor-1)). It has been unexpectedly discovered that Aven interacts with apoptosis regulators including Bcl-2 family members such as Bcl-xL, and Apaf-1 (a mammalian homologue of CED-4, and a facilitator of caspase-9 activation) independently. Aven enhances the anti-apoptotic function of Bcl-xL, against caspase-1 induced apoptosis, as well as protecting cells from apoptosis induced by Apaf-1 and caspase-9. It has been demonstrated that Aven functions as an inhibitor of neuronal apoptosis in an animal model.
The present invention provides an isolated amino acid compound which comprises the amino acid sequence shown in FIG. 1, and functional equivalents thereof. Preferably the amino acid compound has the sequence shown in FIG. 1. The Aven amino acid sequence (SEQ ID NO: 2) shown in FIG. 1 has a Gly/Arg rich amino terminus, a central hydrophobic repeat with predicted alpha-helical structure, and a highly acidic carboxyl terminus. Examples of amino acid fragments which modulate the interaction with BCl-xL include amino acids 71-108 and amino acids 74-104 of FIG. 1. The Aven carboxy-terminus is required for the transcription activation properties of Aven. Deletion of the carboxyl terminus of Aven, from about amino acids 289-362, abolished this transcription activation function.
The invention also provides isolated nucleic acid compounds that comprise a nucleic acid sequence which encodes the presently provided amino acid compounds or parts thereof. Nucleic acid compounds which are DNA are preferred. The most preferred is the DNA compound having the sequence shown in FIG. 2 (SEQ ID NO: 1). Also provided are nucleic acid sequences that are homologous or complementary to any of the provided nucleic acid sequences, and sequences that hybridize to any of the provided nucleic acid sequence.
Expression vectors and host cells overexpressing the amino acid compounds or nucleic acid compounds of the present invention are also provided. The present invention also provides antibodies which bind specifically to Aven, and pharmaceutical compositions comprising substantially purified Aven. In addition, the invention provides agonists and antagonists of Aven. Also provided are stably transformed cell lines expressing the amino acid or nucleic acid compounds of the present invention, or the antibodies, agonists or antagonists of the present invention.
Additional advantages and features of the present invention will be apparent from the following detailed description, drawings and examples which illustrate preferred embodiments of the invention.